Structural Insights Uncover the Specific Phosphoinositide Recognition by the PH1 Domain of Arap3

• Published in: International journal of molecular sciences 2023-01, Vol.24 (2), p.1125
• Main Authors: Zhang, Youjia, Ge, Liang, Xu, Li, Liu, Yongrui, Wang, Jiarong, Liu, Chongxu, Zhao, Hongxin, Xing, Lei, Wang, Junfeng, Wu, Bo
• Format: Article
• Language: English
• Subjects: 1-Phosphatidylinositol 3-kinase; Adaptor Proteins, Signal Transducing - chemistry; Arap3; Biological activity; Breast cancer; complex structure; Crystal structure; Experiments; GTPase-activating protein; GTPase-Activating Proteins - chemistry; Homology; Humans; Hydrogen bonds; Kinases; Lipids; Metastases; Metastasis; Neoplasm Invasiveness; NMR; NMR spectroscopy; Nuclear magnetic resonance; PH domain; Phosphatidylinositol 3,4,5-triphosphate; Phosphatidylinositol Phosphates - metabolism; Phosphatidylinositols; PI(3,4,5)P3; PI(4,5)P2; Pleckstrin; Protein Binding; Protein Domains; Proteins; Recruitment; RhoA protein; Spectroscopy; Surface plasmon resonance
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms24021125

Arap3, a dual GTPase-activating protein (GAP) for the small GTPases Arf6 and RhoA, plays key roles in regulating a wide range of biological processes, including cancer cell invasion and metastasis. It is known that Arap3 is a PI3K effector that can bind directly to PI(3,4,5)P3, and the PI(3,4,5)P3-mediated plasma membrane recruitment is crucial for its function. However, the molecular mechanism of how the protein recognizes PI(3,4,5)P3 remains unclear. Here, using liposome pull-down and surface plasmon resonance (SPR) analysis, we found that the N-terminal first pleckstrin homology (PH) domain (Arap3-PH1) can interact with PI(3,4,5)P3 and, with lower affinity, with PI(4,5)P2. To understand how Arap3-PH1 and phosphoinositide (PIP) lipids interact, we solved the crystal structure of the Arap3-PH1 in the apo form and complex with diC4-PI(3,4,5)P3. We also characterized the interactions of Arap3-PH1 with diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2 in solution by nuclear magnetic resonance (NMR) spectroscopy. Furthermore, we found overexpression of Arap3 could inhibit breast cancer cell invasion in vitro, and the PIPs-binding ability of the PH1 domain is essential for this function.